Today, the majority of new HIV-1 infections result from heterosexual transmission with HIV-1 CCR5-using, R5 strains. However, there is a tight bottleneck during transmission so that only a single virus variant is transmitted in about 80% of infections. It is not known whether the transmitted viruses carry an advantage over strains that fail to transmit. However, R5 envelopes vary extensively in different properties that are likely to have a strong impact on HIV-1 transmission. These properties include (1) macrophage-tropism, (2) capacity to infect cells via low levels of CCR5 and (3) decreased sensitivity to CCR5 ligands including chemokines. Our hypothesis is that HIV-1 transmission is conferred by envelopes with distinct properties that confer an advantage for transmission. We propose the following aims. Aim 1: To investigate whether transmitted founder/acute envelopes form a distinct subset with particular properties: We will investigate HIV-1 R5 clade B and C envs for the three properties described above. We will include envs from the acute stage of infection including founder envelopes that closely represent transmitted strains. We will compare with R5 envelopes from later disease stages that cover the wide variation in the properties to be investigated. Aim 1 experiments will thus reveal whether transmitted founder/acute envelopes form a distinct subset with a particular set of properties. Aim 2: To evaluate whether founder/acute or other R5 envelopes with specific properties confer an enhanced tropism for Langerhan's cells or ectocervical explant cultures as a model for male-to-female transmission: We will investigate the R5 founder/acute envelopes as well as later disease stage envelopes that cover the variation documented in aim 1. These envelopes will be tested for infection of different DC (including LCs) and ectocervical explant cultures and investigated for trans-infection of T-cells via DCs. Together, these experiments will help elucidate how different env properties impact on transmission. Aim 3: To identify the envelope determinants that confer efficient infection of ectocervical explant cultures: We will map envelope determinants that confer efficient infection of ectocervical explant cultures. Our proposal will provide the first comprehensive study of how variation in the properties of R5 envs of different clades impacts on their capacity to infect different DC subsets and ectocervical explant cultures as a model for male-to-female transmission. Importantly, we will identify env properties and determinants that confer efficient transmission. The data obtained will provide new insights into the mechanisms of transmission and help identify vulnerabilities in transmitter envelopes that can be targeted by microbicides and vaccines. PUBLIC HEALTH RELEVANCE: Our application proposes an extensive investigation into the biological properties of HIV-1 viruses that are transmitted. We will use infection of vaginal and cervical cultures as a model of sexual transmission to evaluate which HIV-1 characteristics confer the most efficient infection. Identification of the viral properties and determinants that confer infection of such cultures will provide new insights into the mechanisms of transmission. Our data will help identify vulnerabilities of transmitting viruses that can be targeted by microbicides and vaccines designed to elicit protection against global HIV-1.